In medicine and clinical chemistry, immunoassay techniques are used for the qualitative and quantitative identification of various substances, hereinafter referred to as "ligands", in such body fluids as blood, urine, spinal fluid, amniotic fluid, lymphatic fluid, and the like. Examples of the classes of ligand identified by immunoassay techniques include pharmaceuticals, toxins, drugs of abuse, viral and bacterial antigens, hormones and immunoglobulins.
Most immunoassay techniques can be divided into two classes: radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA and EIA both depend on the competition between sample ligand, that substance suspected of being present in the fluid medium subjected to the assay, and labeled ligand, that same substance taken in a known amount and conjugated to a label or tag enabling one to follow the ligand. The RIA and EIA differ in the type of label employed. In the RIA, a radioisotope is conjugated to the ligand. In the conventional EIA, enzyme is conjugated to the ligand. Although radioisotopes are inherently more sensitive and less subject to serum interference than enzyme labels, several technical and economic factors favor the use of EIA over RIA. These include simplicity, speed, longer shelf life, less expensive instrumentation, avoidance of paperwork, waste disposal, and personnel training required for the use of radioactive materials.
During the past few years, many different EIA systems have been developed. These systems can be divided into two major types: heterogeneous EIA and homogeneous EIA.
Both types of EIA involve the formation of a reaction mixture comprising a minimum of three reaction components: a known amount of ligand, ligand-specific binding component, and sample fluid medium suspected of containing ligand. Heterogeneous EIA involves immobilization of one member of the ligand/ligand-specific binding component pair on solid phase and conjugation of the other member to an enzyme, the label. Typically, ligand is bound to enzyme to form an enzyme-ligand conjugate, the labeled ligand referred to above, and the ligand-specific binding component is immobilized on an insoluble solid phase. The labeled ligand in the enzyme-ligand conjugate then competes with sample ligand suspected to be present in the sample fluid medium for a limited number of ligand-specific binding sites. Once either labeled ligand or sample ligand is bound by the ligand-specific binding component, the bound ligand becomes insoluble. This competition occurs during an incubation step. The insoluble and liquid phases are then separated and the amount of enzyme in each phase quantitated. Sample ligand in the sample fluid medium is determined from the amount of enzyme bound to the solid phase following both these incubation and separation steps. The greater the amount of sample ligand in the sample fluid, the less the amount of enzyme will be found in the insoluble phase. Examples of heterogeneous binding reaction systems in which an enzyme is employed as the label component may be found in U.S. Pat. No. Re 31,006, J. Immunol. Methods, 1:247 (1972), and J. Clin. Microbiol. 3:604 (1976).
The early homogeneous EIA systems similarly employ an enzyme label but bring a simplicity of performance and ease of automation by eliminating the cumbersome separation and pre-incubation steps and by basing enzyme label monitoring on enzyme activity rather than an amount of enzyme bound. Examples of the homogeneous binding reaction systems in which an enzyme is employed as a label component may be found in U.S. Pat. Nos. 3,817,837 and 4,067,774. In these systems, as in the heterogeneous binding reaction systems, ligand is conjugated to enzymes. The formation of the enzyme-ligand conjugate, however, must not result in a change of enzyme activity, the ability of the enzyme to perform its normal catalytic functions. A change in enzyme activity must only occur upon the subsequent binding of the enzyme-ligand conjugate by the ligand-specific binding component. This change in enzyme activity may be an inhibition or stimulation of normal enzyme activity. The presence and quantitation of ligand in the sample fluid is determined by the ability of the sample fluid to protect enzyme activity from the effects of the ligand-specific binding component. Protection is effectuated by the competition between sample and conjugated ligand for ligand-specific binding component binding.
The required retention of enzyme activity following enzyme-ligand conjugation in the enzyme-label systems is, in practice, often difficult to achieve. The heterologous and labile nature of enzymes create problems in characterizing, stabilizing and reproducibly preparing enzyme-ligand conjugates. Additionally, the mandated retention of enzyme activity following ligand-enzyme conjugation restricts the molecular size of the ligand which may be conjugated to enzyme. The size limitation is, in most instances, a stearic one. Ligand must not be so large as to stearically inhibit enzyme activity. These problems have been overcome, to some extent, by replacing the enzyme label with low molecular weight labels monitorable by their respective affects on enzyme activity. These low molecular weight labels, referred to as modulator labels, include prosthetic groups, coenzymes, enzyme substrates, and enzyme modulators. Examples may be found in U.S. Pat. No. 4,238,565; GB Pat. No. 2,023,609A; U.S. Pat. Nos. 4,134,792 and 4,273,866, respectively. The use of the low molecular weight, less than 500 molecular weight, modulator labels, however, has retained the molecular size limitation on the ligands which may be assayed in these systems. As with the enzyme labels, ligand must not be so large as to stearically inhibit the activity of the modulator label to which ligand is conjugated. Thus, the types of ligands assayable in present systems are limited to a molecular size in keeping with the label employed in the assay.
The novel large molecular weight protein label employed in the present invention should greatly expand the molecular size range of ligands which may be assayed for in an EIA. Additionally, since the large molecular weight protein label employed in the present invention is a modulator label, this novel label retains those advantages of other modulator labels such as stability, ease of conjugation and the like. The novel label employed in the present invention is the large molecular weight, biotin-binding, natural inhibitor of pyruvate carboxylase, avidin. Avidin has previously been employed as a ligand-specific binding protein for determination of free biotin in sample fluids. Examples are found in U.S. Pat. No. 4,134,792 and Applied Biochem. & Biotech., 7:443-54 (1982). The novel employment of avidin as a modulator label in the present invention brings with it several advantages inherent in the avidin molecule itself, such as, molecular stability, ease of conjugation, standardization and high yield in conjugate formation.
The present invention also employs a novel use for that class of biotin-containing enzymes of which the preferred enzyme is pyruvate carboxylase. Pyruvate carboxylase is extremely stable, highly active and easily monitored with standard clinical laboratory equipment.
Additionally, pyruvate carboxylase is not present in significant amounts in normal human body fluids such fluids being subjected to the method of the present invention. Thus, unlike some existing EIA systems, pyruvate carboxylase does not cause levels of background interference which must be specifically corrected for.
In a further aspect, the present invention employs a novel enzyme/enzyme modulator combination. The novel combination of biotin-containing enzymes with large molecular weight, biotin-binding enzyme modulators in the present invention provides a novel ligand specific binding assay of the homogeneous or heterogeneous type with enhanced sensitivity, diminished interference, versatility and simplicity of instrumentation.